Methods and compositions for pathogen detection using fluorescent polymer sensors

ABSTRACT

Compositions, methods and related apparatus, as can be used for selective pathogen detection and identification.

This application claims priority benefit from application Ser. No.61/004,471 filed Nov. 28, 2007, the entirety of which is incorporatedherein by reference.

The United States Government has certain rights to this inventionpursuant to Grant Nos. GM077173 and DMI-0531171 from the NationalInstitutes of Health and the National Science Foundation, respectively,to the University of Massachusetts, and Grant No. DE-FG02-04ER46141 fromthe Department of Energy to the Georgia Institute of Technology.

BACKGROUND OF THE INVENTION

Fast and efficient identification of pathogens in water, blood serum andother biological fluids is an important yet unsolved issue in medical,forensic and environmental sciences. Conventional plating and culturingare generally used to identify causative bacterial pathogens in clinicalenvironments. While more technologically advanced systems have beendeveloped for specific microorganisms, these methods are generallycomplex or require sophisticated instrumentation. Plating and culturingis highly accurate, but it is time consuming and requires at least 24 h.Point-of-care treatment decisions are therefore made without havingaccess to crucial microbiological information, often leading to theprescription of a sub-optimal antibiotic. A specific example is thetreatment of keflex- or methicillin-resistant S. aureus strains (MRSA)in community-acquired infections that require prompt treatment witheither sulfa drugs or vancomycin. Researchers investigated>9000 cases ofclinically reported bacterial infections and found that 85-90% were dueto only seven pathogens with S. aureus and E. coli being responsible forhalf of all infections. (B. S. Reisner, G. L. Woods, J. Clin. Microbiol.1999, 37, 2024-2026.) A simple, and rapid test that could discern theclinically most prevalent pathogens (e.g., bacterial, viral, fungal andothers) would be of great value, allowing effective therapeutics againstcausative pathogens to be administered during the initial point-of-carevisit in>85% of all cases. This capability would not only increase theefficacy of therapy, but would also reduce the occurrence ofdrug-resistant bacteria arising from inefficient antibiotics.

Likewise, the detection of bacteria and other pathogens plays a crucialrole in environmental and food safety. For example, E. coli O157:H7 is aworld-wide cause of foodborne illness which is responsible for more than2000 hospitalizations and 60 deaths directly related to thecorresponding bacterial infection each year in the United States, whilethe outbreak in Japan in 1996 resulted in 10000 infections and 11deaths. (P. D. Frenzen, A. Drake, F. J. Angulo, J. Food Prot. 2005, 68,2623-2630; M. D. Disney, J. Zheng, T. M. Swager, P. H. Seeberger, J. Am.Chem. Soc. 2004, 126, 13343-13346.) It has been demonstrated that themajor outbreaks are associated with the contamination of unpasteurizedjuice, vegetables, and water, etc. (P. S. Mead, L. Slutsker, V. Dietz,L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. Griffin, R. V. Tauxe,Emerg. Infect. Dis. 1999, 5, 607-625.) However, testing food forcontamination before consumption is often absent due to the complexand/or lengthy analysis protocols.

SUMMARY OF THE INVENTION

In light of the foregoing, it is an object of the present invention toprovide one or more pathogen detection methods and/or apparatus usedtherewith, thereby overcoming various deficiencies and shortcomings ofthe prior art, including those outlined above. It will be understood bythose skilled in the art that one or more aspects of this invention canmeet certain objectives, while one or more aspects can meet certainother objectives. Each objective may not apply equally, in all itsrespects, to every aspect of this invention. As such, the followingobjects can be viewed in the alternative with respect to any one aspectof this invention.

It can be an object of the present invention to provide, in comparisonwith sensor systems of the prior art, an approach to pathogen detectionand/or identification which is relatively inexpensive, easily preparedand with data quickly processed and analyzed:

It can be another object of the present invention to provide one or moremethods for pathogen detection, to quickly distinguish between bothspecies and strains of pathogens including but not limited to bacteria,viruses, fungi, toxins and other biohazards without resort to markersystems of the prior art.

It can be another object of the present invention, alone or inconjunction with one or more of the preceding objectives, to provide anapparatus and/or kit for ready use in the detection and/oridentification of unknown bacteria or other pathogens.

Other objects, features, benefits and advantages of the presentinvention will be apparent from this summary and the followingdescriptions of certain embodiments, and will be readily apparent tothose skilled in the art having knowledge of various fluorescence-baseddetection methods. Such objects, features, benefits and advantages willbe apparent from the above as taken into conjunction with theaccompanying examples, data, figures and all reasonable inferences to bedrawn therefrom.

In part, the present invention can be directed to a method of detectingthe presence of a bacterium or another pathogen analyte. Such a methodcan comprise providing a non-covalent sensor complex comprising an ionicor otherwise functionalized (e.g., cationic) metal, metallic,semiconductor, metal oxide or other particle component and a polymer orbiopolymer fluorophore component whether ionic, otherwisefunctionalized, or chemically complementary (e.g., anionic) to theparticle component, such a complex having an initial, background orreference fluorescence; irradiating such a sensor complex; andmonitoring an affect and/or change in fluorescence, such monitoring ascan indicate no change, no analyte presence and/or a change notassociated with an analyte of interest, and any such change as can beindicative of the presence of at least one pathogen analyte. In certainembodiments, such a particle component can comprise a hydrophilicmoiety. In certain other embodiments, such a component can comprise ahydrophobic moiety. Regardless, ionic (e.g., cationic) character can beprovided with a quaternary ammonium or other charged group. Thecompositional identity and/or dimension of such a particle component islimited only by pathogen surface interaction. Likewise, the compositionof any such fluorophore component is limited only by complementarychemistry (e.g., anionic) with a particle component, measurablefluorescence and/or change thereof responsive to pathogen contact orinteraction.

Regardless, in certain other embodiments, such a method can comprise aplurality of sensor complexes, each such complex as can provide a changein fluorescence responsive to the presence of a pathogen. As illustratedbelow, such complexes can be varied by fluorophore, particle and/orlinker component, such variations as would be known to those skilled inthe art made aware of this invention. Pathogen interaction can provide afluorescence pattern indicative of the presence of a particular pathogen(e.g., bacteria) species and/or strain.

In part, the present invention can also be directed to a method of usingfluorescent polymer, biopolymer or fluorogenic biopolymer displacementto detect and/or identify pathogen(s). Such a method can compriseproviding a sensor complex of the sort described above, irradiated for atime and/or at a wavelength at least partially sufficient for initialfluorescence; (e.g., background fluorescence as can be due to quenchingby a particle component) contacting such a complex with a pathogenspecies and/or strain, such contact and/or pathogen in an amount atleast partially sufficient to affect fluorescence (e.g., the intensityor wavelength thereof); and monitoring the change in fluorescence uponsuch contact. The sensor complex employed with such a method cancomprise one of those discussed above or illustrated elsewhere herein,alone or in combination with one or more other complexes as can bepresent. Regardless, such a complex can be irradiated at a wavelength atleast partially sufficient for electronic excitement and/or fluorescencethereof. Likewise, as discussed above and illustrated elsewhere herein,contact with such a pathogen can be for a time and/or at a concentrationat least partially sufficient to interact with the particle (e.g.,without limitation metal, metallic, precious metal, metal oxide, sulfideor selenide and/or semiconductor) component of such a complex and/or toaffect fluorescence of the fluorophore component. Such a bacterial orother pathogen species/strain can be present in the context of anunknown sample, the identity of which is limited by competitive and/orpreferential interaction with such a particle component, as compared toparticle component-fluorophore interaction. Alternatively, a pathogencan interact with a polymer component to displace the particle and altercomplex fluorescence. Such a pathogen can be present in the context ofan unknown sample or mixture, the identity of which is limited bycompetitive and/or preferential interaction with such a particle orpolymer component, as compared to particle-fluorophore interaction. Incertain embodiments, the presence of such a pathogen and preferentialinteraction can be observed to enhance fluorescent excited state, as canbe indicated by a change in wavelength or intensity of fluorescence.

In part, the present invention can also be directed to a methoddetecting the presence of and/or identifying one or more unknownpathogen species and/or strains. Such a method can comprise providingreference spectral data comprising change in fluorescence forinteraction of a sensor complex, of the sort described above, with aplurality of reference pathogen species/strains; comparing suchreference data with change in fluorescence for interaction of such asensor complex with unknown pathogens; and identifying the pathogen(s)on the basis of such a comparison. In certain embodiments, suchreference data can comprise fluorescence changes from interaction of aplurality of such complexes with reference pathogen(s). As describedabove, such complexes can be varied by fluorophore component (e.g.,π-conjugation and substitution) and/or fluorescence thereof. Withoutlimitation as to number of sensor complexes employed comprising thereference data, pathogen identification can be made by direct spectralcomparison. Use of a plurality of sensor complexes can provide a patternof fluorescence changes, each such pattern as can be indicative of thepresence of a particular pathogen species and/or strain. Alternatively,comparison can be made using one or more discriminate analysistechniques, as described below.

Alone or in conjunction with discriminate analysis, the presentinvention can also be directed to an apparatus for detection and/oridentification of unknown pathogens. Without limitation as to physicalembodiment or configuration, such a sensor apparatus can comprise amatrix comprising an array of a plurality of sensor complexes of thesort described herein. As illustrated below, such complexes can bechosen to provide differential changes in fluorescence, each such changeresponsive to a wide range of pathogen species and/or strains.Fluorescence change upon pathogen interaction and comparison withreference spectral data, as described above, can be used fordiscriminate pathogen identification.

Likewise, alone or in conjunction with one or more of the methodologiesdescribed herein, the present invention can also be directed to a kitfor detection and/or identification of a bacteria or other pathogen inan analyte sample. Such a kit can comprise one or more nanoparticleswith a coating component on or coupled thereto, such a coating componentas can compromise charged or otherwise interactive terminal groups andone or more fluorophore components, each as described above or as wouldotherwise be understood by those skilled in the art made aware of thisinvention, for non-covalent bonding of one to another. Such a kit canoptionally comprise a fluid medium conducive for pathogen interactionand/or fluorescence. Regardless, such a kit can also comprise a solidmatrix component as can be employed with a plurality of suchnon-covalent sensor complexes and/or unknown pathogen samples.

Without limitation as to methodology, apparatus, kit or applicationcontext, the present invention can be directed to a nano-dimensionedparticulate comprising a core component and a coating component on orcoupled thereto, such a coating component as can comprise charged orotherwise interactive terminal groups. In certain embodiments, such acore component can, without limitation, comprise a metal, a metal oxideand/or a semiconductor material. Notwithstanding core identity, such acoating component can comprise ligands bearing a hydrophilic moiety or ahydrophobic moiety, the latter as can be selected from alkyl,oxa-substituted alkyl and/or poly(alkylene oxide) moieties. Regardless,such moieties can bridge such a terminal group, including but notlimited to quaternary ammonium, and a coupling group including but notlimited to sulfide. The coating component can also comprisepolyelectrolytes including but not limited to polylysine,polyallylamine, polyethyleneimine, and their crosslinked entities. Suchcoatings and/or core components can be selected from those describedmore fully herein or as would be understood by those skilled in the artmade aware of this invention, such selections and/or combinationslimited only by protein interaction of the sort described herein.

Likewise, without limitation as to methodology, apparatus, kit orconjugation with one or more of the aforementioned particulates, thisinvention can be directed to a fluorogenic polymer of a formula

wherein R₁ and R₂ can be moieties independently selected from H, alkyl,oxa-substituted alkyl moieties and/or a moiety sterically configured toat least partially suppress non-specific polymer-pathogen interactions,providing at least one of R₁ and R₂ as such a steric configuration; andR′₁ and R′₂ can be moieties independently selected from charged moietyand counter ion pairs, such a selection at least partially sufficientfor non-covalent interaction of such a polymer component with aparticulate of the sort discussed above; and n can be an integer greaterthan 1 and corresponding to a number of repeating units as can beselected for desired π-conjugation, polymer fluorescence and/or quantumyield, such a component as can be terminated as described herein or aswould be understood by those skilled in the art, depending upon reagentand/or reaction conditions. Without limitation, in certain embodiments,R₁ and R₂ can be independently selected from linear and branchedoxa-substituted alkyl (e.g., poly(alkylene oxide)) moieties and R′₁ andR′₂ can independently comprise carboxylate and/or sulfate groups andcorresponding alkali metal counter ions.

Alternatively, without limitation as to methodology, apparatus, kit orconjugation with one or more of the aforementioned particulates, thisinvention can be directed to a fluorogenic polymer of a formula

wherein R₁ and R₂ can be moieties independently selected from H andinteractive moieties including but not limited to charged moiety andcounter ion pairs, such a selection at least partially sufficient fornon-covalent interaction of such a polymer component with a particulateof the sort discussed above; and n can be an integer greater than 1 andcorresponding to a number of repeating units as can be selected fordesired π-conjugation, polymer fluorescence and/or quantum yield, such acomponent as can be terminated as described herein or as would beunderstood by those skilled in the art, depending upon reagent and/orreaction conditions. Without limitation, in certain embodiments, R₁ andR₂ independently comprise carboxylate and/or sulfate groups andcorresponding alkali metal counter ions. Without limitation, varioussuch fluorogenic polymers are described in a co-pending application,entitled “Methods and Compositions for Protein Detection UsingFluorescent Polymer Sensors,” filed contemporaneously herewith, theentirety of which is incorporated herein by reference.

As illustrated elsewhere herein, other fluorogenic polymers and/orbiopolymers can also be used in conjunction with various particlecomponents, apparatus and/or methods of this invention, such a polymerlimited only by measurable fluorescence and/or change thereof responsiveto pathogen contact or interaction. One non-limiting polymer can begreen fluorescent protein, as described elsewhere herein and/or in theaforementioned incorporated reference. Various otherpolymers/biopolymers useful in the present context would be understoodby those skilled in the art made aware of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B. Design of the nanoparticle-conjugated polymer sensor array.A) Schematic representation of the displacement of anionic conjugatedpolymers from cationic nanoparticles by negatively charged bacterialsurfaces. B) Schematic illustration of fluorescence pattern generationon a microplate. In case of release from the nanoparticle, the initiallyquenched π-conjugated polymers regain their fluorescence. Thefluorescence response is dependent upon the level of displacementdetermined by the relative binding strength of polymer-nanoparticle andbacteria-nanoparticle interactions. By modulating such interactions, thesensor array may generate distinct response patterns against differentbacteria. In the diagram, codes A-G on the microplate represent bacteriaof different types while codes 1-4 denote constructs built up from thefunctional nanoparticles.

FIGS. 2A-B. Receptor and transducer components of the bacterial sensors.a) Structural representation of three cationic gold nanoparticles(NP1-NP3) with various hydrophobic tails. b) Chemical structure of theconjugated polymer (Sw-CO₂) featuring a branched oligo(ethylene glycol)side chain to suppress non-specific polymer-microorganism interactions.

FIG. 3A. Fluorescence titration curves for the complexation of Sw-CO₂(100 nM) with cationic gold nanoparticles (NP1-NP3). The changes influorescence intensity at 463 nm were measured following the addition ofcationic nanoparticles (0-150 nM) with an excitation wavelength of 400nm. The red solid lines represent the best curve-fitting using acalculation model of a single set of identical binding sites.

FIGS. 3B-C. Fluorescence response patterns of nanoparticle-polymerconstructs in the presence of various bacteria (OD₆₀₀=0.05). B)Histograms of fluorescence intensity changes. Each value is an averageof six parallel measurements and the error bars are shown. C)Three-dimensional representation of the fluorescence intensity changesagainst the three nanoparticle-polymer constructs.

FIG. 4. Canonical score plot for the fluorescence response patterns asdetermined with LDA. The first two factors consist of 96.2% variance andthe 95% confidence ellipse for the individual bacteria are depicted.

FIG. 5. Schematic representation of an apparatus comprising anon-limiting nanoparticle and fabrication thereof.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

To address the issue of rapid identification of bacteria, this inventionprovides a protocol for bacterial sensing using an array of goldnanoparticle-conjugated polymer constructs. This sensing protocol adoptsthe concept of the ‘chemical nose’, where a series of analyte receptorsare combined to differentiate targets according to their unique responsediagrams. As shown in FIG. 1, an anionic conjugated polymer is initiallyassociated with cationic gold nanoparticles to affordfluorescence-quenched complexes. In the presence of bacteria, anegatively-charged bacterial surface can competitively interact with thenanoparticles to release the polymer, restoring fluorescence. Withoutlimitation to any one theory or mode of operation, nanoparticles featurea size that seem to enable recognition by extended patches of ahydrophobic or otherwise functionalized microorganism surface. Forexample, poly(L-lysine)-coated gold nanoparticles have been subjected toself-assembly with live bacteria through complementary electrostaticinteractions. The polymer serves to transduce the binding event; theπ-conjugated polymer used in this study provides both multivalency andthe molecular wire effect to facilitate efficient signal generation inthe sensing process. As functional “patches” (e.g. the charged residuesand hydrophobic “hot spots”) are prevalent on cell and microbialexteriors, this strategy has potential applications in theidentification of a wide variety of cells, microbes, and viruses.

To demonstrate the fluorophore displacement strategy an oligo (ethyleneglycol)-substituted carboxylate PPE (Sw-CO₂) and three hydrophobicammonium-functionalized gold nanoparticles (NP1-NP3) were chosen toprovide the sensor elements (FIG. 2). (As indicated above, various otherfluoropolymers can be employed with such nanoparticles or, alternately,with nanoparticles of the sort (e.g., NP1-14) described in theaforementioned incorporated co-pending application.) Fluorescencetitration studies revealed that the cationic gold nanoparticles(NP1-NP3) quench the fluorescence of Sw-CO₂ through formation ofsupramolecular complexes (see FIG. 3A and Table 1). Quenching by thenanoparticle is efficient: typically, an aqueous solution of the polymer(100 nM, based on 12 repeat units/polymer) with a stoichiometric amountof nanoparticle displays approximately 20% of the initial fluorescenceof Sw-CO₂(φ=0.33). The polymer and a stoichiometric amount ofnanoparticles (NP1-NP3) were mixed in 5 mM phosphate buffer (pH 7.4) toyield nanoparticle-Sw-CO₂ constructs with final polymer and nanoparticleconcentrations of 100 nM and 10-40 nM, respectively.

The exposure of these three nanoparticle-Sw-CO₂ constructs towardsbacteria (OD₆₀₀=0.05) induced different levels of fluorescence changes(FIG. 3B). In most cases, the fluorescence of the solution increasesupon addition of the microorganisms. Significantly, the fluorescencechanges exhibit reproducible patterns that depend upon the strains andclasses of bacteria, indicating differentiation in the fluorophoredisplacement. The 12 different bacteria display excellent separationwhen the fluorescence changes were plotted in a three-dimensional graphwith the fluorescence change of the three nanoparticles (NP1-NP3) as therespective axes (FIG. 3C) explicitly demonstrating the ability of theseparticles to discriminate between bacteria.

In selecting nanoparticles that would constitute the sensor array, bothhydrophilic and hydrophobic nanoparticles were initially tested. Uponincubation with bacteria and under the protocols employed, however,hydrophobic nanoparticle constructs (e.g., NP1-NP3) produced significantfluorescence recovery while nanoparticles with hydrophilic tails (e.g.,hydroxyl groups, etc.) induced merely marginal fluorescence changes.Since these two kinds of nanoparticles exhibit comparable bindingaffinity to Sw-CO₂, the difference in the fluorescence recoveryindicates that the former strongly interact with bacteria. Withoutlimitation, a plausible explanation is that the hydrophobic parts of thenanoparticles interact with hydrophobic regions on the surface of thebacteria (e.g. the alkyl chain in teichoic acid), enhancing theelectrostatic nanoparticle-bacteria interaction and, thereby, thefluorescence regeneration. Therefore, both electrostatic and hydrophobicinteractions seem to play important roles in the complexation of theseparticles with bacteria. The engineering of the size, shape andhydrophobicity of the nanoparticle tails would provide a potent means toaugment the diversity in the fluorescence response patterns.

The fluorescence response patterns were analyzed through lineardiscriminant analysis (LDA), a quantitative statistical methodextensively used in pattern recognition. In this method, discriminantfunctions were deduced by maximizing the separation between classesrelative to the variation within classes. As depicted in FIG. 4, LDAtransformed the raw patterns to canonical scores which are clusteredinto 12 groups according to the individual bacteria. The jackknifedmatrix (with cross-validation) in LDA reveals a 100% classificationaccuracy, indicating that such an approach can accurately and reliablydiscern all 12 microorganisms, which contain both Gram-positive (e.g. A.azurea, B. subtilis) and Gram-negative (e.g. E. coli, P. putida)species. The LDA plot does not place the Gram-negative bacteria into aparticular, identifiable part of the graph, suggesting that othereffects are also involved in the discrimination process. Significantly,different strains of E. coli can be easily discerned with the currentsensor array. Again, the three E. coli strains are not groupedparticularly close in the LDA plot, indicating that rather subtledifferences in the bacteria generate marked changes in response.

With patterns of the sort shown in FIG. 3A and Table 2 as the trainingmatrix (3 constructs×12 bacteria×6 replicates), such a sensor array canbe used to identify unknown solutions of bacteria. The unknown sampleswere randomly selected from the 12 bacterial species grown in differentbatches. Fluorescence response patterns generated from the threenanoparticle-polymer constructs was characterized by LDA. Aftertransformation of the unknown patterns to the canonical scores using thediscriminant functions established on the training samples, theMahalanobis distances of the new case to the respective centroids of 12groups were calculated. The closer a specific data set is to the centerof one group, the more likely it belongs to that group. This assignmentis based on the shortest Mahalanobis distance to the 12 bacteria in athree-dimensional space (canonical factors 1 to 3). For the 64 samplesstudied, 61 were correctly identified, resulting in a detection accuracyof>95%. This demonstrates expedient and reliable identification ofbacteria. In particular, the differentiation of the three strains of E.coli suggests that this methodology will be suitable for identificationof pathogenic strains of normally harmless bacteria.

Regardless of the identity of any nanoparticle, fluoropolymer oranalyte, this invention can be embodied by a matrix including an arrayof a plurality of the same or different sensor complexes on, connectedwith and/or coupled to a solid substrate—such as a matrix as can beutilized as or a part of a chip-based sensor, kit or related sensorapparatus for analyte detection and/or identification. For purpose ofillustration only, a representative matrix can be fabricated asillustrated in FIG. 5. For example, a silicon wafer or suitablesubstrate material (e.g., with a hydroxylated surface) can bethiol-functionalized with an appropriate silane reagent, then coupled togold nanoparticles. Optional ligand (e.g., citrate) protection can beremoved and/or exchanged for a cationic ligand component, of the typedescribed herein, for subsequent non-covalent fluoropolymer binding.Microspotter apparatus techniques can be used for surface and/ornanoparticle functionalization and subsequent polymer absorption. Such asurface-based protocol and assembly precludes premixing polymer withparticle. Contact with a fluid medium (e.g., a biofluid possiblycontaining an analyte of interest) can be simply introduced to such achip matrix, with fluorescence and/or change thereof recorded usingeither plate-reader technology or a suitable CCD camera.

As demonstrated with representative, non-limiting embodiments, theintegration of cationic gold nanoparticles with conjugated polymersprovides an easily accessible yet potentially powerful biodiagnostictool, in which the functional nanoparticles and the fluorescent polymerserve as the recognition elements and the transducer, respectively. Theefficient quenching ability of gold nanoparticles coupled with the‘molecular wire’ effect of conjugated polymers compound the pronouncedfluorescence response, which is dictated by the binding strength of thebacterium to the gold nanoparticle. Therefore, manipulating the surfacechemistry of gold nanoparticles and the constitution of the conjugatedpolymer will result in constructs with expanded binding capabilities.Based on the ability to readily differentiate 12 different bacteriausing only three systems, it would be understood in the art that thisinvention can be extended for use in the detection of a wide range ofmicroorganisms including the differentiation of pathogenic and resistantstrains.

EXAMPLES OF THE INVENTION

The following non-limiting examples and data illustrate various aspectsand features relating to the methods and/or articles of the presentinvention, including the detection and identification of unknownpathogen species and strains. In comparison with the prior art, thepresent methods and/or articles provide results and data which aresurprising, unexpected and contrary thereto. While the utility of thisinvention is illustrated through the use of several nanoparticulatesensor complexes and molecular components which can be used to therewithin the context of certain bacterial species/strains, it will beunderstood by those skilled in the art, that comparable results areobtainable with various other nanoparticles and fluorophore componentsin the detection/identification of other pathogens (e.g., viruses,fungi, etc.), as are commensurate with the scope of this invention.

Conjugated polymer (Sw-CO₂) and the cationic gold nanoparticles(NP1-NP3, core diameter˜2 nm) were synthesized according to publishedprocedures. (See, examples 4 and 5 and I. B. Kim, R. Phillips, U. H. F.Bunz, Macromolecules 2007, 40, 5290-5293; C. -C. You, O. R. Miranda, B.Gider, P. S. Ghosh, I. -B. Kim B. Erdogan, S. A. Krovi, U. H. F. Bunz,V. M. Rotello, Nat. Nanotechnol. 2007, 2, 318-323.) The number averagemolecular weight (M_(n)=25 kDa), polydispersity index (PDI=1.8) anddegree of polymerization (P_(n)=12) of Sw-CO₂ were determined by gelpermeation chromatography. The bacterial stocks were donated by Dr. A.Bommarius (Georgia Institute of Technology) and Dr. J. Hardy (Universityof Massachusetts at Amherst): Amycolatopsis azurea (A. azurea),Amycolatopsis orientalis subsp. lurida (A. orientalis subsp. lurida),Bacillus lichenformis (B. lichenformis), Bacillus subtilis (B.subtilis), Escherichia coli (BL21(DE3)) (E. coli (BL2I (DE3)),Escherichia coli (DH5a) (E. coli (DH5a)), Escherichia coli (XL1 Blue)(E. coli (XL1 Blue)), Lactococcus lactis (L. lactis), Lactococcusplantarum (L. plantarum), Pseudomonas putida (P. putida), Streptomycescoelicolor (S. coelicolor), and Streptomyces griseus (S. griseus).

Bacterial cells were grown in LB medium (3 mL) at 37° C. for 16 h to anoptical density of 1.0 at 600 nm. The bacterial cultures werecentrifuged at 4000 rpm for 15 min and washed with phosphate buffer (5mM, pH 7.4) three times. The bacteria were resuspended in phosphatebuffer and diluted to an absorbance of 1.0 at 600 nm. Fluorescenceintensity changes at 463 nm were recorded in 96-well plates (300 μLWhatman® Glass Bottom microplate) on a Molecular Devices SpectraMax M5micro plate reader with an excitation wavelength of 400 nm.

Example 1

Fluorescence titration experiments were conducted to evaluate thecomplexation between nanoparticles and Sw-CO₂. For recording thefluorescence response patterns in the presence of bacteria, Sw-CO₂ andstoichiometric amounts of NP1-NP3, as determined by the fluorescencetitration study (Table 1), were diluted with phosphate buffer (5 mM, pH7.4) to yield solutions with a final polymer (Sw-CO₂) concentration of100 nM. Subsequently, each solution (200 μL) was placed into arespective well on the microplate. After incubation for 15 min, thefluorescence intensity at 463 nm was recorded with an excitationwavelength of 400 nm.

TABLE 1 Binding constants (K_(s)) and binding stoichiometries (n)between anionic polymer (Sw-CO₂) and three cationic nanoparticles(NP1-NP3) as determined from fluorescence titration. NanoparticleK_(S)/10⁸ M⁻¹ −ΔG/kJ mol⁻¹ n NP1 1.12 45.9 2.67 NP2 2.75 48.1 10.0 NP32.71 48.1 6.71

Example 2

Next, 10 μL of a bacterial solution (OD₆₀₀=0.05) was added to each well.After incubation for another 15 min, the fluorescence intensity at 463nm was measured again. The fluorescence intensity before addition ofbacteria was subtracted from that obtained after addition of bacteria,to record the overall fluorescence response (ΔI). This process wascompleted for 12 bacteria to generate six replicates of each, leading toa training data matrix of 3 constructs×12 bacteria×6 replicates (Table2) that was subjected to a classical linear discriminant analysis (LDA)using SYSTAT (version 11.0). The Mahalanobis distances of eachindividual pattern to the centroid of each group in a multidimensionalspace were calculated and the case was assigned to the group with theshortest Mahalanobis distance.

TABLE 2 Training matrix of fluorescence response patterns generated fromNP-(Sw-CO₂) sensor array (NP1-NP3) against various types of bacteria (OD= 0.05 at 600 nm). Bacteria NP1 NP2 NP3 A. azurea 1.320 8.363 7.758 A.azurea 1.875 8.085 11.318 A. azurea 1.643 6.843 7.628 A. azurea 1.8108.313 14.915 A. azurea 1.283 7.735 5.742 A. azurea 1.305 9.253 8.243 A.orientalis subsp. lurida 30.668 32.533 25.598 A. orientalis subsp.lurida 33.398 35.758 34.093 A. orientalis subsp. lurida 31.160 34.17037.205 A. orientalis subsp. lurida 24.005 42.848 29.438 A. orientalissubsp. lurida 29.243 26.558 38.220 A. orientalis subsp. lurida 38.01829.803 41.178 B. lichenformis 195.318 124.438 140.640 B. lichenformis164.993 136.788 142.965 B. lichenformis 163.903 123.355 154.520 B.lichenformis 167.495 120.315 152.213 B. lichenformis 194.945 133.145158.730 B. lichenformis 196.840 125.638 152.393 B. subtilis 235.218174.260 196.053 B. subtilis 232.040 174.323 198.023 B. subtilis 235.505164.763 190.720 B. subtilis 227.188 153.493 185.988 B. subtilis 222.990172.223 189.518 B. subtilis 232.705 156.003 198.215 E. coli (BL21(DE3))17.443 −26.578 −4.338 E. coli (BL21(DE3)) 14.325 −28.983 −3.265 E. coli(BL21(DE3)) 15.175 −18.655 −4.203 E. coli (BL21(DE3)) 16.275 −21.973−2.668 E. coli (BL21(DE3)) 19.198 −20.328 −4.965 E. coli (BL21(DE3))17.968 −19.300 −2.903 E. coli (DH5α) 9.335 61.068 105.800 E. coli (DH5α)11.843 52.365 109.883 E. coli (DH5α) 12.013 60.328 100.815 E. coli(DH5α) 12.360 55.873 92.673 E. coli (DH5α) 9.893 57.170 94.078 E. coli(DH5α) 9.860 70.608 116.055 E. coli (XL1 Blue) 158.553 6.858 105.023 E.coli (XL1 Blue) 171.280 3.700 90.718 E. coli (XL1 Blue) 164.298 9.12094.633 E. coli (XL1 Blue) 177.520 6.280 115.653 E. coli (XL1 Blue)185.140 6.520 128.793 E. coli (XL1 Blue) 178.785 5.023 126.658 L. lactis83.708 80.135 123.985 L. lactis 63.935 97.958 129.270 L. lactis 61.98870.870 145.810 L. lactis 88.515 95.460 133.183 L. lactis 64.880 83.260140.150 L. lactis 87.530 101.368 153.320 L. plantarum −6.428 −15.945−12.628 L. plantarum −7.400 −10.255 −9.813 L. plantarum −3.183 −8.055−8.163 L. plantarum −4.235 −11.848 −13.960 L. plantarum −7.460 −11.235−7.278 L. plantarum −3.730 −16.715 −13.028 P. putida 133.883 77.35884.243 P. putida 138.650 91.008 104.965 P. putida 126.060 62.608 77.063P. putida 145.825 73.970 92.140 P. putida 146.658 71.735 84.733 P.putida 150.738 84.933 109.765 S. coelicolor 45.600 14.303 12.790 S.coelicolor 38.213 20.615 16.215 S. coelicolor 50.170 21.460 14.265 S.coelicolor 46.963 15.608 10.325 S. coelicolor 53.710 10.520 15.943 S.coelicolor 63.685 19.250 21.253 S. griseus 153.198 100.128 147.823 S.griseus 135.543 104.763 142.028 S. griseus 134.793 94.068 173.475 S.griseus 139.033 84.893 162.908 S. griseus 126.708 96.993 143.033 S.griseus 144.808 92.060 166.973

TABLE 3 Accuracy of LDA classification of bacteria analytes (OD₆₀₀ =0.05) from the complexes of the fluorescent polymer (Sw-CO₂) withindividual cationic nanoparticles as sensors. The values are taken fromthe Jackknifed classification matrix based on LDA analysis of the rawdata (6 replicates) listed in Table 2. NP1- NP2- NP3- (NP1-NP3)-Bacteria (Sw-CO₂) (Sw-CO₂) (Sw-CO₂) (Sw-CO₂) A. azurea 100 83 83 100 A.orientalis subsp. 83 100 100 100 lurida B. lichenformis 50 100 33 100 B.subtilis 100 100 100 100 E. coli (BL21(DE3)) 100 100 100 100 E. coli(DH5α) 100 83 33 100 E. coli (XL1 Blue) 50 83 17 100 L. lactis 83 17 50100 L. plantarum 100 100 83 100 P. putida 33 50 67 100 S. coelicolor 6783 83 100 S. griseus 50 67 33 100 Total 76 81 65 100

Example 3

A similar procedure was also performed to identify 64 randomly selectedbacterial samples based on their fluorescence response patterns. Theclassification of new cases was achieved by computing their shortestMahalanobis distances to the groups generated through the trainingmatrix (3 constructs (NP1-NP3)×12 bacteria×6 replicates). During theidentification of unknown bacteria, the bacterial samples were randomlyselected from the 12 respective bacteria and the solution preparation,data collection, and LDA analysis were each performed by differentresearchers, resulting in a double-blind process.

TABLE 4 Identification of 64 unknown bacterial samples with LDA usingassemblies of Sw-CO₂ and NP1-NP3. From the unknown bacterial samples, 61out of 64 were correctly identified, resulting in an accuracy of 95.3%.Correct Fluorescence response Identi- pattern LDA Identificationfication Entry NP1 NP2 NP3 Bacteria Yes/NO 1 142.533 75.388 92.108 P.putida YES 2 182.858 127.943 149.733 B. lichenformis YES 3 77.468 88.975138.883 L. lactis YES 4 30.539 33.934 34.990 A. orientalis subsp. luridaYES 5 18.646 −23.160 −3.736 E. coli (BL21(DE3)) YES 6 11.639 57.813101.611 E. coli (DH5α) YES 7 49.205 16.395 15.680 S. coelicolor YES 8141.730 78.216 92.954 P. putida YES 9 174.401 7.234 109.469 E. coli (XL1Blue) YES 10 31.039 32.559 33.740 A. orientalis subsp. lurida YES 11204.689 153.540 205.669 B. subtilis NO 12 232.956 165.099 192.426 B.subtilis YES 13 1.955 7.644 9.180 A. azurea YES 14 137.791 95.444157.609 S. griseus YES 15 182.769 129.653 151.988 B. lichenformis YES 16−5.669 −12.276 −10.574 L. plantarum YES 17 182.396 124.340 149.264 B.lichenformis YES 18 17.094 −24.721 −4.388 E. coli (BL21(DE3)) YES 1911.456 56.974 104.551 E. coli (DH5α) YES 20 30.545 35.691 32.033 A.orientalis subsp. lurida YES 21 174.821 6.005 113.186 E. coli (XL1 Blue)YES 22 47.008 17.113 15.470 S. coelicolor YES 23 232.094 165.985 191.679B. subtilis YES 24 1.936 9.194 9.234 A. azurea YES 25 11.245 59.013102.789 E. coli (DH5α) YES 26 171.610 5.960 110.973 E. coli (XL1 Blue)YES 27 32.531 32.886 33.358 A. orientalis subsp. lurida YES 28 140.35896.693 155.983 S. griseus YES 29 204.960 155.854 174.626 B. subtilis NO30 13.996 −23.933 −3.560 E. coli (BL21(DE3)) YES 31 47.584 17.030 15.216S. coelicolor YES 32 −4.958 −13.494 −11.673 L. plantarum YES 33 1.4968.318 9.690 A. azurea YES 34 −5.145 −11.691 −10.746 L. plantarum YES 35230.105 165.309 191.754 B. subtilis YES 36 139.821 95.380 156.686 S.griseus YES 37 142.581 75.099 92.426 P. putida YES 38 175.360 6.960113.460 E. coli (XL1 Blue) YES 39 175.956 6.429 110.243 E. coli (XL1Blue) YES 40 177.206 131.679 153.993 B. lichenformis YES 41 1.730 8.0599.379 A. azurea YES 42 143.094 77.610 91.804 P. putida YES 43 176.3457.005 109.099 E. coli (XL1 Blue) YES 44 231.541 165.444 193.734 B.subtilis YES 45 138.209 94.530 155.216 S. griseus YES 46 179.203 126.666151.850 B. lichenformis YES 47 −5.656 −11.515 −11.946 L. plantarum YES48 48.960 17.104 17.126 S. coelicolor YES 49 50.358 16.068 15.233 S.coelicolor YES 50 −4.895 −12.941 −10.746 L. plantarum YES 51 15.855−23.191 −3.371 E. coli (BL21(DE3)) YES 52 48.326 16.870 15.005 S.coelicolor YES 53 10.729 61.713 104.940 E. coli (DH5α) YES 54 15.985−23.040 −3.790 E. coli (BL21(DE3)) YES 55 51.345 17.133 14.724 S.coelicolor YES 56 232.371 166.068 192.690 B. subtilis YES 57 17.031−24.114 −4.018 E. coli (BL21(DE3)) YES 58 11.475 60.711 102.038 E. coli(DH5α) YES 59 1.716 8.224 8.881 A. azurea YES 60 174.838 6.734 115.711E. coli (XL1 Blue) YES 61 1.920 9.241 7.370 A. azurea NO 62 139.83895.484 159.461 S. griseus YES 63 17.225 −24.289 −2.963 E. coli(BL21(DE3)) YES 64 11.541 60.444 102.609 E. coli (DH5α) YES

Example 4 Synthesis of Monomer 5

Synthesis of 1. Triethyleneglycol-monomethylether (200 g, 1.22 mol) wasadded to a dry 1000 mL Schlenk flask. While under N₂ at 100° C., Nametal (10.0 g, 0.440 mol) was added slowly and stirred until all Nametal had reacted. Upon reaction of Na, solution was cooled to 65° C.,and epichlorohydrin (37.0 g, 0.400 mol, 31.4 mL) was added drop-wise.Upon complete addition of epichlorohydrin, reaction mixture was heatedto 100° C., and allowed to react for 3 days. After completion of thereaction, NH₄Cl (0.400 mol, 24.1 g) was added and reacted at 100° C. for1 h. The reaction was cooled to room temperature, filtered to removeexcess salts, and purified by distillation. The first fraction containedexcess starting material, while the second fraction contained product asa light yellowish oil (70.2 g, 0.182 mol, 45.6%).

Synthesis of 2. Compound 1 (15.0 g, 0.0390 mol) was dissolved in dry THF(250 mL). While under N₂, the solution was cooled to 0° C. and NaH (1.2eq, 0.0468 mol, 1.12 g) was added. Upon cessation of H₂, Ts-Cl (0.0390mol, 7.43 g) was dissolved in dry THF (50 mL) and added to the previoussolution. The reaction mixture was warmed to room temperature andreacted overnight. The resulting solution was filtered, the solvent wasremoved, and the crude oil was dried on the pump. The product waspurified by silica gel chromatography (10:1 EtOAc/MeOH). The product wasan orange oil (15.1 g, 0.0279 mol, 71.6%).

Synthesis of 3. A suspension of 1,4-dihydroxy-2,5-diiodobenzene (5.01 g,0.0138 mol), compound 2 (15.0 g, 0.0278 mol), and K₂CO₃ (4 eq., 7.64 g,0.0553 mol) were dissolved in a minimal amount of 2-butanone (75 mL) andheated to reflux. A small amount of KI was added to promote the reactionby exchanging with tosylate. The mixture was reacted for 5 days. Thereaction mixture was cooled to room temperature, diluted with CH₂Cl₂(250 mL), and filtered with celite to remove the black insolubleresidue. The solution was concentrated in vacuo to remove the solventand the crude oil was purified by silica gel chromatography (90:10EtOAc/MeOH) yielding a viscous orange oil (6.20 g, 0.00567 mol, 41.0%).

Synthesis of 4. Compound 3 (4.61 g, 4.11 mmol) was dissolved in dry THF(5 mL) and stirred under N₂ for 15 min. Upon degassing, CuI (0.01 eq.,0.042 mmol, 8.1 mg), (PPh₃)₂PdCl₂ (0.01 eq., 0.042 mmol, 29.5 mg),piperidine (5 mL), and TMS-acetylene (4 eq., 16.8 mmol, 2.39 mL) wereall added to the reaction. The reaction was stirred at room temperaturefor 2 d. The reaction mixture was diluted with THF (25 mL) and filteredto remove any excess salts. The solvent was removed and the crudeproduct was purified by silica gel chromatography (9:1 EtOAc/MeOH). Theresulting product was an orange oil (3.62 g, 3.40 mmol, 83.0%).

Synthesis of 5. Compound 4 (3.62 g, 3.40 mmol) was dissolved in MeOH (50mL). KF (4 eq., 1.07 g, 0.0140 mol) was dissolved in MeOH (25 mL) andadded to the previous solution. The reaction was stirred overnight atroom temperature. The solvent was removed and the crude product wasre-dissolved in CHCl₃. The solution was extracted with H₂O and theorganic fractions were collected and concentrated in vacuo. The crudeproduct was purified by silica gel chromatography (10:1 EtOAc/MeOH)which resulted in an orange oil (2.64 g, 2.96 mmol, 87.1%).

Example 5 Synthesis of Polymer 7

Synthesis of polymer 6: Compound 5 (890 mg, 1 mmol), compound A (534 mg,1 mmol), THF (3 mL), and TEA (3 mL) were all combined in a 25 mL Schlenktube. Upon degassing, (PPh₃)₂PdCl₂ (0.5 mol %, 3.51 mg), and CuI (1 mol%, 1.91 mg) were added to the mixture under N₂ and allowed to react for2 d. The solvent was removed under vacuum and the polymer wasre-dissolved in CHCl₃. The polymer solution was extracted with H₂O (3×),the organic fractions were collected, and the solvent was removed whichresulted in an orange solid (680 mg, 0.60 mmol, 60%). GPC (vs.polystyrene standards in chloroform): M_(n)=25,211, M_(w/M) _(n)=1.837,n=21.

Synthesis of polymer 7: Polymer 6 (660 mg, 0.56 mmol) was deprotected in1 M NaOH and EDTA (250 mg) was added to complex any residual copper. Theresulting solution was neutralized with 1 M HCl, dialyzed against DI H₂Ofor 3 d, and the solvent was removed resulting in a dark orange flakysolid.

Example 6a

As discussed above, various other fluoropolymers can be used inconjunction with present nanoparticles and detection methods. Withreference to FIG. 2A of the aforementioned application,carboxylate-substituted PPE (PPE-CO₂) was synthesized according to aknown procedure. (Bunz, U. H. F. Synthesis and structure of PAEs. Adv.Polym. Sci. 177, 1-52 (2005); Zheng, J. & Swager, T. M. Poly(aryleneethynylene)s in chemosensing and biosensing. Adv. Polym. Sci. 177,151-179 (2005); Kim, I -B, Dunkhorst, A., Gilbert, J. & Bunz, U. H. F.Sensing of lead ions by a carboaxlate-substituted PPE: multivalencyeffects. Macromolecules 38, 4560-4562 (2005).) The weight- andnumber-average molecular weights of the polymer are 6,600 and 3,500,respectively. The polydispersity index and degree of polymerization ofthe conjugated polymer are 1.88 and 12, respectively. Thiol ligandsbearing ammonium end groups were synthesized through the reaction of1,1,1-triphenyl-14,17,20,23-tetraoxa-2-thiapentacosan-25-ylmethanesulphonate with corresponding substituted N,N-dimethylaminesfollowed by deprotection in the presence of trifluoroacetic acid andtriisopropylsilane. Subsequent place-exchange reaction withpentanethiol-coated gold nanoparticles (d≈2 nm) resulted in cationicgold nanoparticles NP1-NP6 in high yields. (See, example 5, below, andBrust, M., Walker, M., Bethell, D, Schiffrin, D. J. & Whyman, R.Synthesis of thiol-derivatised gold nanoparticles in a two-phaseliquid-liquid system. J. Chem. Soc., Chem. Commun. 801-802 (1994).) ¹HNMR spectroscopic investigation revealed that the place-exchangereaction proceeds almost quantitatively and the coverage of cationicligands on the nanoparticles is near unity.

Example 6b Expression and Purification of GFP.

An alternate fluorophoric polymer useful in the context of thisinvention is green fluorescence protein (GFP), which was expressedaccording to standard procedures using E. coli. The Mw and pI of theexpressed GFP is 26.9 KDa and 5.92 respectively. The maximum λ_(ex) andλ_(em) are 490 nm and 510 nm. (See, e.g., FIGS. 6A-B of theaforementioned co-pending incorporated application. Starter culturesfrom a glycerol stock of GFP in BL21(DE3) was grown overnight in 50 mlof 2_YT media with 50 μl of 1000 m ampicilin (16 g tryptone, 10 g yeastextract, 5 g NaCl in 1 L water). The cultures were shook overnight at250 rpm at 37° C. The following day, 5 ml of the starter cultures wasadded to a Fernbach flask containing 1 L of 2_YT and 1 ml1000_amplicilin and shook until the OD₆₀₀=0.7. The culture was theninduced by adding IPTG (1 mM final concentration) and shook at 28° C.After three hours, the cells were harvested by centrifugation (5000 rpmfor 15 minutes at 4° C.). The pellet was then resuspended in lysisbuffer (2 mM Imidizole, 50 mM NaH₂PO₄, 300 mM NaCl). The cells werelysed using a microfluidizer. Once lysed, the solution was pelleted at15000 rpm for 45 minutes at 4° C. The supernatant was further purifiedusing HisPur Cobalt columns from Pierce (cat. Number 89969).

Example 7 Synthesis of Cationic Gold Nanoparticles 4

General procedure: Compound 2 bearing ammonium end groups weresynthesized through the reaction of1,1,1-triphenyl-14,17,20,23-tetraoxa-2-thiapentacosan-25-ylmethanesulphonate (1) with corresponding substituted N,N-dimethylaminesat˜35° C. for 48 h. The trityl protected thiol ligand (2) was dissolvedin dry DiChloroMethane (Methylene Chloride, DCM) and an excess oftrifluoroacetic acid (TFA,˜20 equivalent) was added. The color of thesolution was turned to yellow immediately. Subsequently,triisopropylsilane (TIPS,˜1.2 equivalent) was added to the reactionmixture. The reaction mixture was stirred at room temperature for ˜5 hunder Ar atmosphere. The solvent and most TFA and TIPS were distilledoff under reduced pressure. The pale yellow residue was washedthoroughly with hexanes and further dried in high vacuum. The productformation was quantitative and their structure was confirmed by ¹H NMR.Subsequent place-exchange reaction of compound 3 dissolved in DCM withpentanethiol-coated gold nanoparticles (d˜2 nm) was carried out atambient temperature for 3 days. Then, DCM was evaporated under reducedpressure. The residue was dissolved in a small amount of distilled waterand dialyzed (membrane MWCO=1,000) to remove excess ligands, acetic acidand the other salts present with the nanoparticles. After dialysis, theparticles were lyophilized to afford a brownish solid. The particles areredispersed in deionized water (18 MΩ-cm). ¹H NMR spectra in D₂O showedsubstantial broadening of the proton signals and no free ligands wereobserved.

Example 8a Synthesis of Alternate Ligands

General procedure: Compound II bearing ammonium end groups weresynthesized through the reaction of1,1,1-triphenyl-14,17,20,23-tetraoxa-2-thiapentacosan-25-ylmethanesulphonate (I) with corresponding substituted N,N-dimethylaminesat ˜35° C. for 48 h. The trityl protected thiol ligand (II) wasdissolved in dry DiChloroMethane (Methylene Chloride, DCM) and an excessof trifluoroacetic acid (TFA,˜20 equivalents) was added. The color ofthe solution was turned to yellow immediately. Subsequently,triisopropylsilane (TIPS,˜1.2 equivalents) was added to the reactionmixture. The reaction mixture was stirred for ˜5 h under Ar condition atroom temperature. The solvent and most TFA and TIPS were distilled offunder reduced pressure. The pale yellow residue was washed thoroughlywith hexanes and further dried in high vacuum. The product (L) formationwas quantitative and their structure was confirmed by ¹H NMR. The yieldswere>95%.

Compound L1: ¹H NMR (400 MHz, CDCl₃, TMS): δ 3.95 (br, 2H, —CH₂N—),3.70-3.58 (m, 14H, —CH₂O—+—OCH ₂—(CH₂N)—), 3.49 (t, 2H, —CH₂O—), 3.25(s, 9H, —N(CH₃)₃), 2.90 (s, 3H, —CH₃SO⁻ ³⁻), 2.52 (q, 2H, —CH₂S—),1.64-1.51 (m, 4H, (SCH₂)CH ₂+—CH ₂(CH₂O)—), 1.36-1.22 (m, 15H,—SH+—CH₂—).

Compound L2: ¹H NMR (400 MHz, CDCl₃, TMS): δ 3.94 (br, 2H, —CH₂N—),3.69-3.56 (m, 14H, —CH₂O—+—OCH ₂—(CH₂N)—), 3.44 (t, 2H, —CH₂O—),3.40-3.32 (m, 2H,—NCH₂—), 3.23 (s, 6H, —(CH₃)₂N—), 2.78 (s, 3H, —CH₃SO⁻₃—), 2.51 (q, 2H, —CH₂S—), 1.69-1.149 (m, 4H, (SCH₂)CH ₂+—CH ₂(CH₂O)—),1.44-1.24 (m, 18H, —SH+—CH₂—+—(NCH₂)CH ₃).

Compound L3: ¹H NMR (400 MHz, CDCl₃,TMS): δ 3.95 (br, 2H, —CH₂N—),3.68-3.56 (m, 14H, —CH₂O—+—OCH ₂—(CH₂N)—), 3.46 (t, 2H, —CH₂O—),3.40-3.33 (m, 2H,—NCH₂—), 3.19 (s, 6H, —(CH₃)₂N—), 2.87 (s, 3H, —CH₃SO⁻₃—), 2.52 (q, 2H, —CH₂S—), 1.76-1.53 (m, 6H, —(NCH₂)CH ₂—)+(SCH₂)CH₂+—CH ₂(CH₂O)—), 1.41-1.22 (m, 21H, —SH+—(NCH₂CH ₂—)+—CH₂—), 0.89 (t,3H, —CH₃—).

Compound L4: ¹H NMR (400 MHz, CDCl₃, TMS): δ 3.95 (br, 2H, —CH₂N—),3.81-3.72 (m, 1H, H_(Cyclo)), 3.69-3.53 (m, 14H, —CH₂O—+—OCH ₂—(CH₂N)—),3.49 (t, 2H, —CH₂O—), 3.11 (s, 6H, —(CH₃)₂N—), 2.91 (s, 3H, —CH₃SO⁻ ₃—),2.52 (q, 2H, —CH₂S—), 2.23 (d, 2H, H_(Cyclo)), 1.99 (d, 2H, H_(Cyclo)),1.78-1.52 (m, 4H, —(SCH₂)CH ₂+—CH ₂(CH₂O)—), 1.51-1.12 (m, 21H,SH+—CH2—+H_(Cyclo)).

Compound L5: ¹H NMR (400 MHz, CDCl₃, TMS): δ 8.37 (d, 1H, H_(Ar)), 7.98(d, 1H, H_(Ar)), 7.69-7.61 (m, 3H, H_(Ar)), 7.59-7.48 (m, 1H, H_(Ar)),4.38 (br, 2H,—NCH₂—Ar)), 3.76 (br, 2H, —CH₂N—) 3.72-3.62 (m, 14H,—CH₂O—+—OCH ₂—(CH₂N)—), 3.61-3.55 (m, 2H, —CH₂O—), 3.23 (s, 6H,—(CH₃)₂N—), 3.07 (s, 3H, —CH₃SO⁻ ₃—), 2.52 (q, 2H, —CH₂S—), 1.67-1.51(m, 4H, —(SCH₂)CH ₂+—CH₂(CH₂O)—), 1.35-1.21 (m, 15H, —SH+—CH₂—).

Compound L6: ¹H NMR (400 MHz, CDCl₃, TMS): δ 3.94 (br, 2H, —CH₂N—),3.75-3.52 (m, 16H, —CH₂O—+—OCH ₂—(CH₂N)—+—CH ₂—OH), 3.48 (t, 2H,—CH₂O—), 3.39-3.31 (m, 2H,—NCH₂—), 3.25 (s, 6H, —(CH₃)₂N—), 3.2 (br, 1H,—OH), 2.89 (s, 3H, —CH₃SO⁻ ₃—), 2.52 (q, 2H, —CH₂S—), 2.35-2.26 (m, 2H,—(NCH₂)CH ₂—), 1.70-1.52 (m, 4H, +(SCH₂)CH ₂+—CH ₂(CH₂O)—), 1.36-1.21(m, 15H, —SH+—CH₂—).

Compound L7: ¹H NMR (400 MHz, CDCl₃, TMS): δ 4.78 (br, 1H,—CHOH(CH2OH)—), 4.59 (br, 1H, —CH2OH—), 4.50-4.45 (m, 1H,—CHOH(CH2OH)—), 4.43 (d and br, 2H, —NCH₂—), 3.95 (d and br, 2H,—CH2N—), 3.86-3.76 (d and br, 2H, —CH ₂—OH), 3.75-3.55 (m, 14H,—CH₂O—+—OCH ₂—(CH₂N)—), 3.48 (t, 2H, —CH₂O—), 3.34 (s, 6H, —(CH₃)₂N—),2.99 (s, 3H, —CH₃SO⁻ ₃—), 2.52 (q, 2H, —CH₂S—), 1.71-1.51 (m, 4H,+(SCH₂)CH ₂+—CH ₂(CH₂O)—), 1.42-1.21 (m, 15H, —SH+—CH₂—).

Compound L8: ¹H NMR (400 MHz, CDCl₃, TMS): δ 3.96 (br, 2H, —CH₂N—),3.79-3.75 (m, 1H, H_(Cyclo)), 3.66-3.57 (m, 14H, —CH₂O—+—OCH ₂—(CH₂N)—),3.46 (t, 2H, —CH₂O—), 3.12 (s, 6H, —(CH₃)₂N—), 2.89 (s, 3H, —CH₃SO⁻ ₃—),2.52 (q, 2H, —CH₂S—), 2.28 (d, 2H, H_(Cyclo)), 2.01 (d, 2H, H_(Cyclo)),1.64-1.54 (m, 4H, —(SCH₂)CH ₂+—CH ₂(CH₂O)—), 1.47 (q, 2H, H_(Cyclo)),1.33 (t, ³J=8.0 Hz, 1H, —SH), 1.30-1.22 (m, 14H, —CH2—), 1.16 (q, 2H,H_(Cyclo)) 1.04 (td, 1H —CHC—), 0.86 (s, 9H, —C(CH₃)₃—).

Compound L9: ¹H NMR (400 MHz, CDCl₃, TMS): δ 7.82 (d, 2H, H_(Ar)),7.66-7.51 (m, 3H, H_(Ar)), 4.24 (br, 2H, —CH₂N—), 3.78 (s, 6H,—(CH₃)₂N—), 3.68-3.52 (m, 14H, —CH₂O—+—OCH ₂—(CH₂N)—), 3.47-3.36 (m, 2H,—CH₂O—), 2.87 (s, 3H, —CH₃SO⁻ ₃—), 2.52 (q, 2H, —CH₂S—), 1.70-1.46 (m,4H, —(SCH₂)CH ₂+—CH ₂(CH₂O)—), 1.42-1.1.16 (m, 15H, —SH+—CH₂—).

Compound L10: ¹H NMR (400 MHz, CDCl₃, TMS): δ 3.98 (br, 2H, —CH₂N—),3.78-3.75 (m, 1H, H_(Cyclo)), 3.64-3.55 (m, 14H, —CH₂O—+—OCH ₂—(CH₂N)—),3.46-3.42 (dt, 2H, —CH₂O—), 3.16 (s, 6H, —(CH₃)₂N—), 2.86 (s, 3H,—CH₃SO⁻ ₃—), 2.52 (q, 2H, —CH₂S—), 1.93-1.40 (m, 26H, SCH₂)CH ₂+—CH₂(CH₂O)—+H_(Cyclo)), 1.33 (t, ³J=7.82 Hz, 1H, —SH), 1.29-1.24 (m, 14H,—CH2—).

Compound L11: ¹H NMR (400 MHz, CDCl₃, TMS): δ 7.4-7.2 (m, 4H, H_(Ar)),7.17 (d, 1H, H_(Ar)), 3.95 (d and br, 2H, —CH₂N—), 3.79-3.52 (m, 14H,—CH₂O—+—OCH ₂—(CH₂N)—), 3.45 (q, 2H, —CH₂O—), 3.29-3.22 (m and br, 1 H,H_(Cyclo)), 3.01-2.92 (m and br, 1H, H_(Cyclo)) 2.87 (s, 3H, —CH₃SO⁻₃—), 2.81 (d and br, 6H, —(CH₃)₂N—), 2.52 (q, 2H, —CH₂S—), 2.39-2.26 (m,2H, H_(Cyclo)), 2.19-2.06 (m, 2H, H_(Cyclo)), 1.96-1.84 (m, 4H,H_(Cyclo)), 1.72-1.53 (m, 4H, —(SCH₂)CH ₂+—CH ₂(CH₂O)—), 1.42-1.1.19 (m,15H, —SH+—CH₂—).

Compound L12: ¹H NMR (400MHz, CDCl₃, TMS): δ 7.42 (d, 2H, H_(Ar)),7.37-2.27 (m, 8H, H_(Ar)), 7.25-7.18 (t, 2H, H_(Ar)), 5.13 (s, 1H,H_(Ar)), 4.12 (br, 2H, —CH₂N—)), 3.96 (br, 2H, —NCH ₂(CH₂OCAr),3.64-3.51 (m, 14H, —CH₂O—+—OCH ₂—(CH₂N)—), 3.45 (t, 2H, —CH₂O—),3.29-3.34 (m, 2H, —CH2OCAr—), 3.28 (s, 6H, —(CH₃)₂N—), 2.86 (s, 3H,—CH₃SO⁻ ₃—), 2.52 (q, 2H, —CH₂S—), 1.60-1.48 (m, 4H, —(SCH₂)CH ₂+—CH₂(CH₂O)—), 1.34-1.16 (m, 15H, —SH+—CH₂—).

Compound L13: ¹H NMR (400 MHz, CDCl₃, TMS): δ 3.96 (br, 2H, —CH₂N—),3.72 (s, 1H, —(CH₃)₂NCH—), 3.70-3.53 (m, 14H, —CH₂O—+—OCH ₂—(CH₂N)—),3.46 (t, 2H, —CH₂O—), 3.33 (s, 6H, —CH(OCH ₃)₂), 3.28 (s; 6H,—(CH₃)₂N—), 2.89 (s, 3H, —CH₃SO⁻ ₃—), 2.51 (q, 2H, —CH₂S—), 1.69-1.53(m, 4H, (SCH₂)CH ₂+—CH ₂(CH₂O)—), 1.40-1.23 (m, 15H, —SH+—CH₂—+—(NCH₂)CH₃).

Example 8 b

Synthesis of Ligand L14

Procedure: Compound IV bearing L-Phe group was synthesized through thereaction of1,1,1-triphenyl-14,17,20,23,26-pentaoxa-2-thiaoctacosan-28-oic acid(III) with corresponding2-amino-N-(2-(dimethylamino)ethyl)-3-phenylpropanamide. Briefly,compound III was dissolved in a mixture of dry DCM and DMF that wasplaced in an ice-bath. When the temperature reached about 0° C.,corresponding L-phenylaline derivative,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),N-hydroxybenzotriazole (HOBt), and sodium bicarbonate were added. Themixture was stirred at room temperature for 24 h. Subsequently, thesolution was poured into water and extracted with ethyl acetate (EtOAc).The organic layers were combined and washed successively with saturatedsodium bicarbonate and brine. After drying over sodium sulfate, thesolvent was removed under reduced pressure. The residue was charged onSiO₂ column for purification. EtOAc/MeOH (90:10) and EtOAc/MeOH/NH₄OH(90:10:1) were used as gradient eluent. Compound V was obtained throughnucleophilic substitution of compound IV with bromoethane. The tritylprotected thiol ligand (V) was dissolved in dry DCM.TFA and TIPS wereadded successively. The reaction mixture was stirred at room temperaturefor ˜5 h. Subsequently, the solvent was removed under reduced pressure.The residue was washed thoroughly with diethyl ether to remove theresidual TFA and TIPS. After drying in high vacuum, the product L14(5-benzyl-N-ethyl-32-mercapto-N,N-dimethyl-4,7-dioxo-9,12,15,18,21-pentaoxa-3,6-diazadotriacontan-1-aminium)was obtained in quantitative yield. Its structure was confirmed by ¹HNMR.

Compound L14: ¹H NMR (400 MHz, CDCl₃, TMS): δ 8.48 (br t, 1H, —NH—),7.65 (br d, 1H, —NH—), 7.25 (m, 5H, H_(Ar)), 4.61 (m, 1H, —CH<), 4.03(q, 2H, —OCH₂—), 3.8 ˜3.4 (m, 22H, —OCH₂—+—CH₂—), 3.14 (m, 2H, —CH₂Ar),3.11 (s, 6H, —CH₃), 2.90 (m, 2H, —CH₂—), 2.52 (q, 2H, —SCH₂—), 1.58 (m,4H, —CH₂—), 1.26 (m, 17H, —CH₂—+—CH₃).

Fabrication of Cationic Gold Nanoparticles.

General procedure: 1-Pentanethiol coated gold nanoparticles (d=˜2 nm)were prepared according to a previously reported protocol. (Brust, etal., J. Chem. Soc. Chem. Commun., 1994, 801.) Place-exchange reaction ofcompound Ls dissolved in DCM with pentanethiol-coated gold nanoparticles(d˜2 nm) was carried out for 3 days at ambient temperature. (See,Hostetler, et al., Langmuir, 1999, 15, 3782.) Then, DCM was evaporatedunder reduced pressure. The residue was dissolved in a small amount ofdistilled water and dialyzed(membrane MWCO=1,000) to remove excessligands, acetic acid and the other salts present with the nanoparticles.After dialysis, the particles were lyophilized to afford a brownishsolid. The nanoparticles are redispersed in deionized water (18 MΩ-cm).¹H NMR spectra in D₂O showed substantial broadening of the protonsignals and no free ligands were observed.

As demonstrated through several non-limiting embodiments, non-covalentconjugates of gold nanoparticles and a fluorescent polymer identifybacteria effectively within minutes. With this protocol,nanoparticle-bacteria interactions release an initially boundfluorescent polymer from the gold nanoparticle quencher, resulting in a“turn-on” of the polymer fluorescence. The unique fluorescence responseswhich are generated by the conjugates of different nanoparticles withbacterial surfaces provide an efficient means of differentiation. Theefficacy of this method as shown by using 12 different bacteria,demonstrating ability to differentiate between species of bacteria aswell as between differing strains of a single species, without the useof antibodies or radioactive markers.

1-27. (canceled)
 28. A composition comprising a plurality ofnon-covalent complexes between a plurality of nanoparticles and afluorescent polymer, wherein each of the plurality of nanoparticlescomprises an inner metallic core and a coating layer comprising acationic ligand, wherein the fluorescent polymer comprises an anionicgroup, and wherein fluorescence of the polymer in the non-covalentcomplexes is quenched.
 29. The composition of claim 28, wherein theplurality of non-covalent complexes comprise at least three non-covalentcomplexes between at least three different nanoparticles and afluorescent polymer.
 30. The composition of claim 28, wherein thecationic ligand comprises a quaternary ammonium ion.
 31. The compositionof claim 28, wherein the fluorescent polymer is a synthetic 7-conjugatedpolymer.
 32. The composition of claim 31, wherein the syntheticπ-conjugated polymer comprises a structural unit of:

wherein R₁ and R₂ are independently selected from H, alkyl andoxa-substituted alkyl groups; R′₁, and R′₂ are independently selectedfrom H and alkyl groups; provided that at least one of R′₁ and R′₂comprises a charged group.
 33. The composition of claim 32, wherein atleast one of R′₁, and R′₂ comprises a carboxylate or a sulfate anion anda counter cation.
 34. The composition of claim 33, wherein each of R′₁,and R′₂ is:


35. The composition of claim 32, wherein at least one of R₁, and R₂comprises a poly(alkylene oxide) group.
 36. The article of claim 35wherein each of R₁, and R₂ is:


37. A composition comprising a plurality of non-covalent complexesbetween a nanoparticle and a plurality of fluorescent polymers, whereinthe nanoparticle comprises an inner metallic core and a coating layercomprising a cationic ligand, wherein each of the fluorescent polymerscomprises an anionic group, and wherein fluorescence of the fluorescentpolymers in the non-covalent complexes is quenched.
 38. The compositionof claim 37, wherein the plurality of non-covalent complexes comprise atleast three non-covalent complexes between a nanoparticle and at leastthree different fluorescent polymers.
 39. The composition of claim 37,wherein the coating layer of the nanoparticle has covalently bondthereon a cationic ligand having the structure of

wherein R is selected from:


40. The composition of claim 37, wherein at least one of the fluorescentpolymers is a synthetic π-conjugated polymer.
 41. The composition ofclaim 40, wherein each of the π-conjugated polymers comprises astructural unit of:

wherein R₁ and R₂ are independently selected from H, alkyl andoxa-substituted alkyl groups; provided that at least one of R₁ and R₂comprises a charged group.
 42. The composition of claim 41, wherein atleast one of R₁, and R₂ comprises a carboxylate or a sulfate anion and acounter cation.
 43. The composition of claim 42, wherein each of R₁, andR₂ is:


44. The composition of claim 37, wherein at least one of the fluorescentpolymers is a natural fluorescent protein.
 45. The composition of claim44, wherein the natural fluorescent polymer comprises a greenfluorescent protein.
 46. A composition useful for detecting the presenceof a pathogen analyte, comprising a composition of any of claims
 28. 47.An assay for detecting the presence of a pathogen analyte, comprisingcomposition of any of claims 28.